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To evaluate the effect of WNT7A in vivo, PDX studies were conducted. Four cases of fresh tumor tissues were obtained from patients undergoing surgery, which were provided by the First Affiliated Hospital of Fujian Medical University (Approval Number: FJMU-IACUC 2021-0299) and the written informed consent of each participant was obtained. The collected tumor tissues were cut into small fragments (2–3 mm in size) and placed in DMEM. Then 2–3 mm³ tumor blocks were implanted into the male nude mice (BALB/c nu/nu, 6–8 weeks old, Gempharmatech, China). When the primary mouse tumor grew to about 100 mm³, it was transplanted into the second generation. Two cases were chosen based on the second-generation expression of WNT7A, and transplanted into 6 mice of each group. Once the PDX tumors reach a suitable size or when the study endpoint is reached, the mice are euthanized, and the tumors are harvested for further analysis. Based on the formula below, we calculated the volume of the tumor: volume = length × width² × 0.5.

The use and handling of animals were reviewed and approved by the Ethics Committee for Animal Experiments of Jinan University. All experiments were approved by the Animal Research and Use Committee of Jinan University. The details were the same as those of the tumor xenograft experiments presented in our previous studies (Li et al., 2017 (link)). Nude mice (BALB/C nu/nu, 4–6 weeks old, female) reared under standardized conditions were purchased from GemPharmatech Co., Ltd. (Nanjing, Jiangsu, China). Huh7 cells in equal volumes of PBS and Matrigel were implanted subcutaneously into the flanks of nude mice. When the volume of the tumors reached approximately 50 mm³, the mice were randomly divided into three groups (n = 5) and treated with intraperitoneal injection of either PBS or maprotiline (20 mg/kg, 40 mg/kg) twice a week for three weeks. The mice were sacrificed after the treatment course, the tumors and blood for further tests were collected and properly stored. Cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), red blood cells (RBCs), white blood cells (WBCs), neutrophils, lymphocytes, hemoglobin (HGB) and platelets (PLTs) were measured by Servicebio Biotechnology Company (Wuhan, China).